Our past work on this grant directly addressed immune aspects of Periodontal Disease (PD) by development of methods for isolation of gingival mononuclear cells (GMC) from the disease site. We have shown that large numbers of immunoglobulin (Ig) secreting, spot forming cells (SFC) occur, which are mainly restricted to IgG-subclass (IgG1>IgG2>IgG3=IgG4) and less so to IgA-subclass (IgA1>IgA2) responses. This unique system has allowed us to determine the degree of antigen- specific SFC responses in PD, and we showed that Porphyromonas gingivalis fimbriae and LPS are major and minor components of these IgG- and IgA- subclass responses, respectively. Thus, our work suggests that the predominant IgG- and IgA-plasma cell responses seen in PD have both polyclonal and antigen-specific components. This has logically led to an analysis of cytokines present in GMC supernatants (sups), which would account for these unique profiles of B cell responses. Our studies have now shown that IL-6 is a major terminal differentiation factor in GMC sups, together with IL-5. Of importance to this proposal is the presence of a potentially novel cytokine which induces IL-6 receptor (IL-6R) expression on human lymphocytes. We plan to continue these exciting studies by addressing the precise pattern of cytokines present in individual cell types in GMC which account for B cell and plasma cell responses. We will focus on IL-2, IL-4, IL-5, IL-6 and IFN-gamma and use both PCR and in situ hybridization for these cytokines in both immune (T helper (Th) cells subsets, B cells, monocytes/macrophages) and nonimmune (fibroblasts and epithelial cells) cells from tissues of patients with PD. Our preliminary studies suggest that gingival Th cells may be induced, in part, by the presence of P. gingivalis fimbriae acting as a superantigen and by heat shock proteins (hsps). We plan to assess CD4+ Th cells from GMC for responses to these antigens and to determine if hsps induce Th1-type (IFN-gamma and IL-2) and fimbriae (superantigen) elicits mainly Th2 (IL-4, IL-5 and IL-6) responses. By specific derivation of cloned CD4+ Th cells, we can determine the relative contribution of Th1 and Th2 cells for inflammation and B cell responses, respectively. The potential role of TGF-beta and IL-4 in switches to IgG- and IgA-subclasses will be addressed. We will determine if the IL- 6R inducing factor is a novel cytokine, and if so, we will purify and clone this factor in order to evaluate its role in B cell responses. Finally, we plan to determine the relative contribution of antigen- specific T cells and derived cytokines for B cell responses, with emphasis on fimbriae and hsps. this comprehensive analysis of Th cells and cytokines will provide the cellular and molecular basis for B cell responses in human PD and may be of predictive importance in classification of the different forms of this disease.